tbuckley/cabot-search huggingface dataset:
CSIG-22. CAMKIIΓ INTERACTS WITH HIF-1Α IN AN OXYGEN-INDEPENDENT MANNER TO DRIVE PLASTICITY IN GLIOBLASTOMA
Authors: Divya Ventarapragada, Graysen McManus, Weidong Zhou, Cheol Park, Killian Buechler, Amelia Stepniak, Jason Miska, Atique Ahmed
Journal: Neuro-Oncology (2023)

Abstract Glioblastoma (GBM) is a highly aggressive brain tumor that demonstrates treatment resistance. Hypoxia-inducible factor 1-alpha (HIF-1α) plays a crucial role in GBM progression and recurrence. While HIF-1α overexpression is correlated with poor GBM survival, clinical inhibitors have shown limited efficacy due to compensatory pathways that are not well understood. In previous studies, we discovered GBM upregulates HIF-1α after temozolomide (TMZ) through an unknown mechanism independent of physiological hypoxia. We demonstrated HIF-1α but not HIF-2α knockdown reduces the stem-like properties of GBM and enhances their sensitivity to TMZ therapy in vitro and in vivo. To elucidate the mechanism of TMZ-induced HIF-1α stabilization, we performed co-immunoprecipitation of HIF-1α followed by mass spectrometry analysis (IP-MS). Our findings revealed 99 binding partners that were significantly and uniquely upregulated after TMZ treatment compared to GBM cells cultured under normal oxygen tension (20% O2), hypoxia (1% O2), or DMSO. Among these interactions, we focused on the HIF-1α-CAMKIIγ axis for further investigation based on pathway enrichment, patient survival data, and co-expression analysis in the TCGA_GBM dataset. Co-immunoprecipitation experiments confirmed enhanced binding between HIF-1α and CAMKIIγ in different GBM patient-derived xenograft (PDX) lines while this interaction was significantly reduced in normal astrocytes. Knockdown of CAMKIIγ inhibited the transcriptional activity of HIF-1α, increased its protein turnover, and improved TMZ sensitivity compared to control cells following TMZ treatment. Additionally, the expression of stem-cell markers and neurosphere formation, which are associated with GBM aggressiveness, were attenuated in hypoxia and TMZ conditions when CAMKIIγ was absent. Our data demonstrated that the HIF-1α-CAMKIIγ axis is critical for TMZ-induction of HIF signaling and cellular plasticity.
CTIM-33. EFFICACY AND SAFETY STUDY OF NEOADJUVANT EFINEPTAKIN ALFA (NT-I7) AND PEMBROLIZUMAB IN RECURRENT GLIOBLASTOMA
Authors: Mason Webb, Ugur Sener, Terry Burns, Erin Twohy, Sani H Kizilbash, Michael Ruff, Joon Uhm, Evanthia Galanis, Stacy D'Andre, Cecile Riviere-Cazaux, Byung Ha Lee, Lynn Flickinger, Ian Parney, Jian Campian
Journal: Neuro-Oncology (2023)

Abstract Glioblastoma (GBM) is the most common and aggressive form of brain tumor in adults. Despite maximal surgical resection, irradiation, and chemotherapy, median overall survival (OS) for recurrent GBM (rGBM) remains at only 30 weeks. GBM tumors have an immune-suppressive microenvironment with low numbers of tumor-infiltrating lymphocytes (TILs). Interleukin 7 (IL-7) plays a key role in T cell homeostasis and survival, with IL-7 inducing T cell expansion in lymphopenic conditions. Efineptakin alfa (NT-I7), a long-acting IL-7, has been shown to increase effector CD8 T cells and reduce suppressor T-regulatory cells in murine GBM models, with subsequently improved OS. We recently completed a Phase I study in newly diagnosed GBM patients which showed that NT-I7 is safe, well-tolerated, and increased absolute lymphocyte counts (ALCs), and determined maximum tolerated dose for Phase II. We hypothesized that NT-I7 may not only mitigate GBM treatment-associated immune suppression, but also potentiate the effect of immune checkpoint blockade, leading to an increase in TILs and improved survival. The purpose of this study is to evaluate the safety and efficacy of combining NT-I7 and pembrolizumab in rGBM patients. This is an open-label, single arm one-stage Phase II study of NT-I7 and pembrolizumab as neoadjuvant and adjuvant therapy with surgery for adult patients with rGBM. We plan to enroll 30 evaluable patients, including a 6-patient safety run-in. The primary endpoint is the 9-month OS rate. Secondary endpoints include progression-free survival, objective response rate, changes in ALC, and adverse events profile. The correlative endpoints include studies to assess the anti-glioma immune response, changes in T cell subsets and functionality, peripheral cytokine quantification, and immunohistochemical analysis of GBM tissue. Longitudinal cerebrospinal fluid proteomics is optional. This study opened in Jan 2023. Six (6) of the planned thirty (30) patients had been enrolled by time of submission.
TMIC-27. THE ROLE OF AQUAPORIN-4 IN GLIOBLASTOMA-ASSOCIATED VASOGENIC CEREBRAL EDEMA
Authors: Tanvi Joshi, Dolores Hambardzumyan
Journal: Neuro-Oncology (2023)

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Vasogenic cerebral edema is a severe complication that occurs in a majority of GBM patients, leading to a significant increase in intracranial pressure, accompanying neurological deficits, and increased mortality rates. Edema in GBM patients is almost exclusively managed by the corticosteroid dexamethasone (DEX), which has recently been found to have several drawbacks, including immunosuppression and interference with radiation therapy. Thus, there is an urgent need to better understand the mechanisms of edema resolution in GBM in order to identify alternative treatment strategies. The water channel aquaporin-4 (AQP4) is a critical player in the brain's water homeostasis, with increased levels in GBM compared to the normal brain. To gain more insight into the role of AQP4 in GBM-associated edema, we used the RCAS/tv-a system to generate de novo GBM with different driver mutations in Aqp4 knockout (KO), heterozygous (HET), and wild-type (WT) mice. Multiplex IF staining combined with RNAScope of tumors showed that AQP4 expression co-localized with GFAP-positive reactive astrocytes in perivascular areas. Our results indicated that tumor-bearing mice in an Aqp4-deficient background had increased edema content and decreased survival, suggesting that AQP4 is important for edema resolution from tumors. Loss of Aqp4 also significantly decreased astrocyte coverage in the tumor core, but not in the tumor periphery. Moreover, anti-VEGFA treatment only improved vessel leakage and reduced edema in WT tumor-bearing mice. Ongoing studies are focused on characterizing the impact of Aqp4 loss on the gene expression profile of tumor core-specific astrocytes using single-cell RNA sequencing and spatial transcriptomics. We are also studying the cellular distribution of AQP4 in GBM and the effect of DEX treatment on AQP4 levels.
CTIM-28. FIRST-IN-HUMAN TRANSGENE DETECTION 17 MONTHS POST-ADENOVIRAL THERAPY
Authors: Maria Varela, Molly West, Syed M Faisal, Andrea Comba, Maria Castro, Pedro Lowenstein
Journal: Neuro-Oncology (2023)

Abstract Gene therapy was developed as a promising approach to treat high-grade glioma (HGG). One of the main strategies is suicide gene therapy. This method introduces the expression of an enzyme that converts a non-toxic prodrug to an active toxic compound, thereby causing the death of transduced cells. Specifically, herpes simplex virus thymidine kinase (HSV1-TK) converts the pro-drug valacyclovir into phosphorylated valacyclovir. When cells initiate DNA replication, phosphorylated valacyclovir is incorporated into de novo DNA strands, causing DNA replication to arrest, followed by apoptosis. This approach is lethal only for replicating cells, (i.e., tumor cells), without harming non-dividing brain cells. An infected cell, expressing HSV1-TK, requires two further simultaneous events to induce cell death: (1) to be exposed to valacyclovir and (2) enter into cell division. Without either of these events, the cell will remain alive and will still express HSV1-TK. Previous work from our lab in murine models demonstrated the long-term expression of transgenes β-galactosidase and HSV1-TK up to 12 months after viral therapy administration (Zermansky et al 2001; Barcia et al 2007). We recently completed and published our Phase I clinical trial (NCT01811992), where dual vector viral therapy, Ad-TK/Ad-Flt3L, and valacyclovir was administered to patients suffering from HGG (Umemura et al, 2023). In paraffin embedded tumor samples from recurrent tumors to our surprise we detected HSV1-TK immunoreactivity up to 17 months after initial treatment. HSV1-TK immunoreactive cells were found in five out of eight patients’ samples. We analyzed HSV1-TK expression to further characterize HSV1-TK positive cells using combination immunohistochemistry, allowing us to identify them as tumor cells, neurons, or astrocytes. The presence of HSV-TK positive cells in the recurrent tumors strongly supports the administration of further cycles of pro drug (valacyclovir), following Ad-TK administration.
TMIC-25. NEUTROPHIL-INDUCED GLIOBLASTOMA FERROPTOTIC CELL DEATH DEPENDS ON LC3-ASSOCIATED PHAGOCYTOSIS OF NEUTROPHILS BY GLIOBLASTOMA CELLS
Authors: Tong Lu, Patricia Yee, Jessica Thorpe, Hong-Gang Wang, Wei Li
Journal: Neuro-Oncology (2023)

Abstract Glioblastoma multiforme (GBM) is the most aggressive and common type of primary adult brain cancer. Tumor necrosis is a histological hallmark of GBM and a poor prognostic factor for the disease. Although it is thought to result from chronic ischemia, the molecular mechanisms that induce tumor necrosis remain unclear. Previously, we reported that tumor-associated neutrophils (TANs) contribute to the generation of tumor necrosis through inducing ferroptosis in GBM cells. We showed that accumulation of lipid peroxides within GBM cells depends on neutrophil myeloperoxidase (MPO). The cell killing is associated with transfer of MPO-containing granules to GBM cells. (Yee et al., 2020, Nature Communications, DOI: 10.1038/s41467-020-19193-y). During further study of Neutrophil-induced glioblastoma ferroptotic cell death, we found that neutrophil engulfment by GBM cells is required for intercellular transfer of neutrophil contents and the upregulation of lipid peroxides in GBM cells. The engulfment process can be inhibited by RGDS peptides, which inhibit the function of RGDS-containing integrins. Furthermore, we used immunofluorescence imaging to show that internalized neutrophils colocalize to molecular components of the LC-3 associated phagocytosis (LAP) machinery. Disruption of these components either through pharmacological or genetic means rescues GBM cells from neutrophil-induced cell death. We then used an orthotopic xenograft GBM mouse model to demonstrate that disruption of the LAP machinery in tumor cells reduces tumor necrosis and improves survival. Altogether, this study identified a novel ferroptosis-inducing process, which can help to understand how neutrophils contribute to tumor necrosis in GBM. Additionally, the results suggested that targeting LAP may have therapeutic benefits for GBM treatment.
TMIC-83. THE MOLECULAR AND SPATIAL ARCHITECTURE OF AN IMMUNE-RICH GLIOMA SUBTYPE
Authors: Anupam Kumar, Lin Wang, David Solomon, Shawn Hervey-Jumper, Mitchel Berger, Aaron Diaz, Joanna Phillips
Journal: Neuro-Oncology (2023)

Abstract Diffuse glioma exhibit an immunosuppressive microenvironment, yet glioma subsets driven by RAF-alterations exhibit an increased immune signature. In pleomorphic xanthoastrocytoma (PXA), we identified CXCL14 as an important factor promoting a cytotoxic T cell response. The molecular and spatial relationships within this tumor type, however, are unknown. To address this, we use single nucleus RNA sequencing (snRNAseq), spatial transcriptomics, and spatial molecular imaging to construct a high-resolution molecular landscape of PXA. SnRNAseq was performed on 36,781 cells from 10 tumors, including initial and recurrent pairs, spatial transcriptomes were performed on a total of 91 FOVs comprised of 17 PXAs, and spatial molecular imaging (SMI) of 1,000 RNA probes at subcellular resolution was performed on 48,518 cells from 6 tumors, including two PXA, two pilocytic astrocytoma, and two ganglioglioma. For snRNAseq, neoplastic cells were separated from non-malignant cells based on analysis of inferred copy number alterations. CellChat was used to infer cell-cell communication. For spatial transcriptomics, data from 200-300µm FOVs were normalized and divided into CD3-high versus CD3-low groups. For SMI, cells with fewer than 20 transcripts were eliminated, data were normalized using simple total counts, cells were segmented using the Nanostring pipeline, and semi-supervised clustering was performed. Dual CXCL14 RNAscope and Iba1/CD3 immunostaining confirmed spatial relationships within the tumor. In PXA with high-CD3, differential gene expression analysis demonstrated broad immune-enrichment and identified upregulation of several ligand-receptor pairs. Integrating these data with snRNAseq and cell-cell communication analysis inferred paracrine interactions between tumor cells and T cells. One such factor, CXCL14, was expressed widely by tumor cells but spatial analysis revealed cohesive clusters of CXCL14-high expressing tumor cells. In anaplastic (CNS WHO grade 3) tumors, however, both CXCL14 expression and microglia decreased. These high-resolution molecular analyses of PXA identify dynamic tumor-immune crosstalk regulating the immune microenvironment.
TMIC-34. SKULL-DERIVED MYELOID CELLS AUGMENT T CELL RESPONSES AND SUPPRESS GLIOBLASTOMA GROWTH
Authors: Meeki Lad, Venina Kalistratova, Jacob Young, Sabraj Gill, Atul Saha, Jennifer Rios, Diana Salha, Austin Lui, Manish Aghi
Journal: Neuro-Oncology (2023)

Abstract While recent studies have implicated the skull marrow as a functional immune reservoir in CNS pathology, its contribution to malignancy remains unexplored. To that end, we transplanted fluorescently-labeled calvarial flaps into syngeneic glioblastoma-bearing mice and cytometrically characterized tumor-infiltrative immune cells, observing a robust population of skull-derived myeloid cells (SDMCs; 40.3% of all intratumoral myeloid cells) in a Balb/C mouse tumor model (CMFDA-stained flaps, 72h). These SDMCs were robust, remaining viable for 3 weeks in an alternative longitudinal model (C57BL/6 mice, GFP+ flaps). As MHCII+ leukocytes were disproportionately enriched among SDMCs (87.9% vs 67.3%, n=3, p=0.012), we interrogated the immunogenicity of these cells through targeted skull ablation, achieved via 13 Gy irradiation of body-shielded mice prior to orthotopic glioblastoma implantation. Indeed, skull-ablated mice had reduced infiltration by antigen-presenting cells than controls (cDCs: 1.1% vs 4.5%, p=0.002; macrophages: 14.4% vs 36.6%, n=10/group, p&amp;lt; 0.001) and 6-fold greater necrosis (67.7% vs 13.5% apoptotic, p&amp;lt; 0.001), suggesting an impaired ability to mount a controlled, adaptive immune response. To clarify the role of SDMCs in mediating secondary immune function, we administered the CXCR4 antagonist AMD3100 between the inner and outer tables of the skull, inhibiting marrow homing signals and promoting myeloid precursor egress (0.26% vs 0.02% of intratumoral myeloid cells, n=10/group, p&amp;lt; 0.001). This yielded cDC2 expansion (10.4% vs 3.0%, p=0.003) and consequent CD4+ T effector memory differentiation (92.3% vs 60.0%, p=0.007). These differences were clinically meaningful, as skull irradiation accelerated tumor growth and attenuated survival compared to controls, while AMD3100 treatment accomplished the opposite (median OS: 17d vs 22d, p&amp;lt; 0.001; 26.5d vs 22d, p=0.006). Concurrent αCD4/CD8/Thy1.2 administration abrogated the therapeutic effect of AMD3100 entirely, confirming the dependency of SDMC-mediated tumor suppression on T cells. Together, our work clarifies the antitumoral role of skull marrow and represents the first-ever therapeutic modulation of this space.
CTIM-31. CLINICAL IMPACT OF ADDING PEMBROLIZUMAB TO STANDARD OF CARE CHEMORADIATION IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA
Authors: Prakash Ambady, Ramon Barajas, Rochelle Fu, Laszlo Szidonya, Amy Huddleston, Lauren Popp, Dana Moussalli, Maria Turchan, Claire Diaz, Neuwelt Edward
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND Standard of care (SOC) for newly diagnosed glioblastoma includes radiotherapy plus temozolomide followed by 6 adjuvant cycles monthly temozolomide per the Stupp protocol. We report the secondary objectives of a single arm phase 2 study evaluating the clinical impact of the addition of immune check point inhibitor pembrolizumab to this standard therapy. METHODS Patients with histopathology proven glioblastoma (WHO 4) eligible to receive SOC were prospectively enrolled to received pembrolizumab 200 mg iv every 3 weeks for up to 35 doses, progression, development of dose limiting toxicity or withdrawal of consent. Patients also receive ferumoxytol steady state MRI scans at baseline, 4 weeks after the last day of standard of care stereotactic radiosurgery or chemoradiotherapy, every 9 weeks thereafter until suspected radiographic progression, and then within 4 weeks from suspected radiographic progression. Primary endpoints of the study were to test the sensitivity and specificity of ferumoxytol steady state imaging for response assessment in this cohort. Secondary objective includes Progression free (PFS) and Overall survival (OS) and toxicity. PFS and OS were analyzed using Kaplan-Meier method. RESULTS A total of 52 patients (19 F, 33 M; 43 IHD wt and 9 IDH mu; 25 MGMT gene methylated, 27 unmethylated) were enrolled and included. At a median Follow up of 14.3 months, median PFS was 10.7 m (95% CI:8.1-12.6) and median OS was 14.4 m (95%CI: 12.5-16.9). Two grade 3 or higher toxicities (bullous dermatitis and Steven-Johnson syndrome) were attributed to pembrolizumab. CONCLUSIONS Although, the addition of pembrolizumab to SOC was well tolerated in most patients, this study did not see any meaning clinical benefit in PFS or OS with the addition of pembrolizumab in patients with newly diagnosed glioblastoma. Analysis for the primary imaging endpoint is ongoing.
TMET-09. SEX DIFFERENCES IN SERINE-DERIVED GLYCINE BIOSYNTHESIS DRIVE RESPONSES TO COMBINED NUTRIENT DEPRIVATION AND PHARMACOLOGICAL INHIBITION IN GBM
Authors: Jianyun Lu, Camryn Marquez, Jasmin Sponagel, Prakash Chinnaiyan, Joshua B Rubin, Joseph Ippolito
Journal: Neuro-Oncology (2023)

Abstract Glioblastoma (GBM) is the most common and deadly primary brain malignancy. Male GBM patients occur at higher incidence and display more aggressive disease than females. Ambiguity of molecular mechanisms driving sexually dimorphic outcomes in GBM limits the implementation of clinically effective treatments. Therefore, our group seeks to determine and target the metabolic bases of sex-biased GBM proliferation and progression. We have identified glucose utilization as a unique determinant of male but not female GBM patient outcomes. Utilizing a mouse model for mesenchymal GBM (Nf1-/-DNp53), we demonstrated a male-specific decrease in proliferation in response to glucose-deprived media and glycolytic inhibition. Following labeling with [13C] glucose, males had significantly higher de novo serine biosynthesis, measured by enrichment of serine and glycine. This was confirmed with significant upregulation of several serine biosynthetic pathway enzymes in male cells. These mouse model data were validated by metabolomics and enzyme data from human GBM and lung cancer, including pan-cancer gene expression data, underscoring the broad translational importance of these findings. We then sought to determine if sex-biased responses to serine and glycine deprivation were similar to those observed following glucose deprivation. Utilizing serine and glycine-deprived media conditions, we identified a male-specific decrease in proliferation following pharmacological inhibition of the serine biosynthetic pathway. More precisely, in the absence of exogenous glycine, we discovered that pharmacological inhibition of de novo synthesis of glycine from serine drove male-specific decreases in proliferation. Additionally, we discovered that under serine and glycine-deprived conditions, inhibition of de novo synthesis of glycine from serine sensitized female transformed astrocytes to antifolates. These data highlight that de novo glycine synthesis drives sex differences in GBM cell proliferation. As well, our work suggests that dietary restriction of serine and glycine, in combination with pharmacological inhibition of de novo glycine biosynthesis, represents a novel GBM treatment paradigm.
TMET-11. BERBERINE AS A POTENTIAL ENHANCER FOR 5-ALA-MEDIATED FLUORESCENCE IN GLIOBLASTOMA: INCREASING DETECTABILITY OF INFILTRATING GLIOMA STEM CELLS TO OPTIMIZE 5-ALA-GUIDED SURGERY
Authors: Satoshi Suehiro, Yoshihiro Ohtsuka, Akihiro Inoue, Takanori Ohnishi, Daisuke Yamashita, Masahiro Nishikawa, Hideaki Watanabe, Takeharu Kunieda
Journal: Neuro-Oncology (2023)

Abstract OBJECTIVE The prognosis of glioblastoma multiforme correlates with residual tumor volume. In fluorescence-guided surgery, 5-aminolevulinic acid (5-ALA) has been used to maximize safe resection. We searched for repositioning drugs that affect mitochondrial functions and energy metabolism, identifying berberine (BBR) as a potential enhancer of 5-ALA-mediated protoporphyrin IX (PpIX) fluorescence (5-ALA-fluorescence). Here, we investigated whether BBR can be applied to clinical practice as a 5-ALA-fluorescence enhancer. METHODS Effects of BBR on 5-ALA-fluorescence in glioma cells and GSCs were evaluated by flow cytometry (fluorescence-activated cell sorting; FACS) analysis. As 5-ALA is metabolized for heme synthesis, effects of BBR on mRNA expressions of seven enzymes in the heme-synthesis pathway were analyzed. Enzymes showing significantly higher expression than control in all cells were identified and protein analysis was performed. To examine clinical availability, the detectability and cytotoxicity of BBR in tumor-transplanted mice were analyzed. RESULTS Fluorescence microscopy revealed much more intense 5-ALA-fluorescence in both GSCs and non-stem cells with 5-ALA and BBR than with 5-ALA alone. FACS disclosed that BBR greatly enhanced 5-ALA-fluorescence compared to 5-ALA alone and enhancement was much higher for GSCs than for glioma cells. Among the seven enzymes examined, BBR upregulated mRNA expressions of ALA synthetase 1 (ALAS1) more highly in all cells, and activated ALAS1 through deregulating ALAS1 activity inhibited by the negative feedback of heme. In vivo study showed that 5-ALA-fluorescence with 5-ALA and BBR was significantly stronger than with 5-ALA alone, and the sensitivity and specificity of BBR-enhanced fluorescence were both 100%. In addition, BBR did not show any cytotoxicity for normal brain tissue. CONCLUSIONS BBR enhanced 5-ALA-mediated PpIX fluorescence by upregulating and activating ALAS1 through deregulation of negative feedback inhibition by heme. BBR is a clinically used drug with no side effects. BBR is expected to augment fluorescence-guided surgery and photodynamic therapy.